DE4423392A1 - Methods of identifying antigenic peptides - Google Patents

Abstract

The invention concerns in vitro methods of identifying and producing antigen peptides and peptide mixtures which simulate in vivo trimming of foreign or the body's own proteins; and the use of the antigen peptides or peptide mixtures identified and produced by the claimed method, for example, for identifying vaccine components against pathogens or tumour cells, or competitive or antagonistic peptides in auto-immune diseases or in the production of vaccines.

Description

The invention relates to in vitro methods for identification tion of antigenic peptides that process in vivo Simulate foreign or proprietary protein and use the antigen identified by the method according to the invention NEN peptides, for example for the identification of vaccine building blocks against pathogens or tumor cells or from compe titive or antagonistic peptides in autoimmune diseases units.

One of the most important basic principles of the body's own defense reactions is the presentation of processed foreign egg knows through the body's own polymorphic structures, the so-called tissue tolerance antigens (major histocompatibility major histocompatibility complex antigens MHC antigens). Present as peptides from the MHC molecules Processed foreign or proprietary proteins are processed by two different types of lymphocytes with different ones Functions recognized in the immune system, the so-called cytotoxi T-lymphocytes and the helper T-lymphocytes.  

Cytotoxic T lymphocytes (= Cytotoxic T lymphocytes = CTLs) play a significant role in both defense against intracellular viral and bacterial infections as well against degenerate cancer cells. You recognize antigens in form of small peptides that bind to class I MHC antigens the surface of infected or degenerated cells they are. The peptides recognized by the defending CTLs are in the cell during the normal cell cycle continuous, proteolytic degradation of the antigen proteins generated.

Helper T lymphocytes also play a central role in the body's defense. For example, you practice one Function in the proliferation and differentiation of anti gene-recognizing B cells. Helper T cells recognize pep tide localized by cells presenting on antigen MHC class II molecules are presented. The one from the Peptides recognized by T helper cells are mainly exogenous nen, d. H. non-body origin.

A very important role in the generation of antigenic peptides in the cell presented by MHC class I molecules play multienzyme complexes called proteasomes (A.L. Goldberg and K.L. Rock, Proteolysis, proteasomes and antigen presentation, Nature 357 (1992), 375). These proteasomes can come from the cytosol of cells be isolated.

The assembly of the antigenic peptides with MHC class I moles cooling takes place in the endoplasmic reticulum (ER), after the Transport of the peptides through the ER membrane instead. Involved on the transport of the peptides into the ER and the assembly with MHC class I molecules are members of the "ABC Transporter "protein family (Higgins, ABC transporters: from microorganisms to man. Annu. Rev. Cell Biol. 8 (1992), 67), called "Transporter for Antigen Presentation" (TAP) (J.J.  

Monaco, A molecular model of MHC class-I-restricted antigen processing, immunol. Today 13 (1992), 173; J.J. Monaco, S. Cho and M. Attaya, Transport protein genes in the murine MHC: possible implications for antigen processing, Science 259 (1990), 1723; M.J. Androlewicz, K.S. Anderson and P. Cresswell, Evidence that transporters associated with antigen processing translocate a major histocompatibility complex class I-binding peptides into the endoplasmic reticulum in an ATP-dependent manner., Proc. Natl. Acad. Sci. USA 90 (1993), 9130; J.C. Shepherd, T.N. Schumacher, P.G. Ashton-Rickardt, S. Imaeda, H.L. Ploegh, C. Janeway Jr. and S. Tonegawa, TAP1-dependent peptide translocation in vitro is ATP dependent and peptide selective, Cell 74 (1993), 577; J.J. Neetjes and F. Momburg, Cell biology of antigen presentation. Curr. Opin. Immunol. 5 (1993) 27). Of the Assembling the peptides with the MHC molecules is a selek tive process and requires that the peptide a certain Size (9 ± 1 amino acids) (H.G. Rammenee, K. Falk and O. Rotzschke, Peptides naturally presented by MHC class I molecules. Annu. Rev. Immunol. 11: 213 (1993). Farther must have certain "anchor amino acids" in the molecular pockets of the Bind MHC class I molecules. For different MHC molecules individual, allele-specific anchor motifs were determined (H.G. Rammenee, K. Falk and O. Rotzschke, MHC molecules as peptide receptors. Curr. Opin. Immunol. 5 (1993), 35; K. R.O. Falk, S. Stevanovic, G. Jung, H-G. Ramsee, alleles specific motifs revealed by sequencing of self-peptides Eluted from MHC Molecules, Nature 351 (1991), 290). With With the help of these anchor motifs, antigenic peptides from Prote Viruses, bacteria, parasitic protozoa and tumor cells predicted and identified (H.J. Pamer, M.J. Bevan, Precise Prediction of a Dominant Class I MHC- restricted Epitope of Listeria monocytogenes, Nature 353 (1991) 852; H.J. Wallny D.K., S. Faath, G. Jung, A. van Pel, T. Boon, H-G. Rammenee Identification and quantification of a Naturally Presented Peptide as  Recognized by Cytotoxic T Lymphocytes Specific for an Immunogenic Tumor Variant, Int. Immunol. 4: 1088 (1992).

The processing route of the MHC class II molecules Antigenic peptides presented are similar in wide areas that of MHC class I molecules present molecules. Particularities arise in particular in that the mostly exogenous proteins pass through the endocytic pathway fen, for presentation a length of about 12 amino acids must have and in special compartments, the so-called called compartments for peptide loading (compartments for peptide loading = CPL) with MHC class II molecules asso adorn after this early endosomal process have gone through the path and prevent the peptide loading changing invariant chain has been reduced (Schmid & Jackson, Nature 369 (1994), 103-104 and Re cited therein conferences; Germain and Margulies, Annu. Rev. Immunol. 11 (1993), 403-450).

After assembling the MHC-peptide complexes, they turn on transports the cell surface to the immune system to get presented.

The number of "naturally processed and presented Peptides "(NPP) that have been identified to date is re relatively small and limited to a small spectrum various peptides from Pro expressed in large quantities teine. The identification of antigenic determinants based on due to low expression rate, poor processing, slow transport into the ER or suboptimal binding MHC molecules, in very small amounts on the cell surface area presented is available with the previously available standing methods, even using highly sensitive ven methods, such as tandem mass spectrometry, difficult to impossible. The general, experimentally empirical confirmed rule that all peptides generated in the cell are transport into the ER and around available free MHC molecules  compete also applies to peptides that are made from proteins pa organisms or cancer cells. This fact che makes it extremely difficult in very small quantities existing antigenic peptides from a large excess isolate dogeneric peptides that contain most of the MHC Occupy molecules.

Just the identification of these rare antigenic peptides is of great importance for vaccine development and immunotherapy for many diseases. The present The invention is therefore based on the object of a method to provide with these rare, on MHC molecules ge bound, antigenic peptides can be identified.

The solution to this problem is given in the claims featured embodiments provided.

The invention thus relates to a method for identification antigenic peptides comprising the following steps:

• (a) Proteolytic degradation through cellular processing mechanisms of exogenously added proteins or polypep tiden that contain the antigenic peptides to a pep tide size that allows association with MHC molecules;
• (b) Association of the peptides obtained in step (a) with suitable MHC molecules;
• (c) isolation of the MHC molecule-peptide complexes;
• (d) separation of the antigenic peptides from the MHC molecules and
• (e) Determination of the amino acid sequence of the in step (d) holding antigenic peptides.

Antigenic peptide here means a peptide that is recognized by the immune system as a hapten.

A peptide size that is associated with MHC molecules leaves, for MHC class I molecules it is one with 9 ± 1 Amino acids, while they are about 12 in MHC class II molecules Amino acids.  

Under cellular processing mechanisms here sol understood that of cytosolic multienzyme complexes (= Proteasomes) and others, sometimes not yet purely Darge Enzymes / enzyme complexes are exercised as well as the subsequent processing ("trimming") in microsomes.

The MHC molecule-peptide complexes are isolated before preferably from microsome or CLP lysates.

To facilitate the identification of antigenic peptides that generated by the cellular processing machinery and are presented by MHC molecules on the cell surface, the stoichiometric ratio between endogenous Pep tid and antigenic peptides are manipulated so that the antigenic peptides in a large excess for the trans port into the ER and for binding to MHC molecules for ver stand by. This is due to the addition of exogenous Pro tein or polypeptide reached in step (a). At the same time should be the amount of endogenous peptides already in the ER bound to MHC molecules are small. That means more free MHC molecules for the antigenic peptides are available.

Compared to the previously used methods and procedures of Peptide elution from cell lysates or from affini purified MHC molecules, can be described by the here bene procedures a strong enrichment of specific, Pathogen-derived peptide epitopes can be achieved. This enables subdominant peptide epitopes to be identified adorn the under normal physiological conditions too little concentration in MHC-bound form to the Cell surface get to specific CTL progenitor cells to induce expansion and differentiation.

Dominant epitopes often represent proteins or protein sections that are in large quantities from the pathogen are formed (e.g. envelope proteins of viruses), but not absolutely under such a high functional selection pressure stand as that they cannot be changed within certain limits  are. This leads to z. B. in an infection with Hu man immunodeficiency virus (HIV) most immune responses against protein segments that are very variable are. The dominant epitopes cover the sub dominant epitopes in MHC-bound presentation. There at is the concentration of subdominant epitopes on the Cell surface so small that it does not act as a signal to the dif differentiation of the T cells to CTLs is sufficient.

Because basically all proteins come from the cellular machine processed and presented, is invented by the inventor Process according to the invention a high enrichment of pep tidepitopen reaches that under normal conditions subdo are minant. If you can generate CTLs against epitopes that Represent peptide sections that are under such high radio tional selection pressures that they are a very limited possess limited genetic variability, one can with Using such CTLs to produce a very effective immune response Once activated, CTLs also recognize subdomi nant epitopes that were found in the lowest concentrations cells are presented, whereas the border concentration tion for the activation of T cells and differentiation CTLs is much higher.

Many viruses owe their persistence to this fact because the dominant epitopes constantly mutate and thus the virus ins total of immune elimination, despite constantly being present Immune reactions, deprived. The method according to the invention can thus advantageously be used to identify viral antigenic peptides used for prophylaxis and Immunotherapy against such viruses can be used.

The method according to the invention enables antigenic pep isolate tide from cellular MHC molecules in quantities, that are several orders of magnitude above the quantities that can be achieved with methods known in the prior art can. Antigenic peptides can be made directly from complete  Microsome lysates, cell lysates or from affinity-purified loaded, MHC molecules can be obtained.

In a preferred embodiment of the invention The MHC molecules are MHC class I molecules.

Suitable MHC molecules are those molecules that have a pas Send conformation to pre antigenic peptides to be able to present.

In a further preferred embodiment of the invented According to the inventive method, the proteolytic degradation takes place Proteasomes for proteins or polypeptides.

This embodiment of the method according to the invention is particularly preferred if the peptides from the degradative Degradation of the proteins or polypeptides by MHC class I moles cooler should be presented. The proteasomes are before preferably according to that of Boes et al., J. Exp. Med. 179 (1994), Method described 901 isolated. Conveniently the protein with the isolated proteasomes in microsome Standard buffer (Levy et al., Cell 67 (1991), 265) for below different periods (0.5 to 96 hours) at 37 ° C incu beer. The source of proteasomes is preferably the cell line K-562 strongly expressing this enzyme complex (ATCC No. CCL 243).

In a further preferred embodiment of the invented The MHC class I molecules originate from the method according to the invention from cellular microsomes. Isolation of the microsomes preferably follows that of Saraste et al., Proc. Natl. Acad. Sci. USA 83 (1986), 6425.

In a further preferred embodiment of the invented The method according to the invention is associated with the antigen NEN peptides with the MHC class I molecules by incubation of peptides with microsomes.

The unloaded MHC class I molecules are mainly in of the microsome fraction (Levy et al., op. cit.).  

As a result, it is advantageous to use the Proteasome-Ab build obtained antigenic peptides directly with the microsomal To incubate fraction of the endoplasmic reticulum To obtain MHC class I molecule-antigenic peptide complexes.

Incubation is preferably from 1 minute to 36 hours at 37 ° C.

Another preferred embodiment of the invention hits a process where the MHC molecules are MHC class II molecules are.

A particularly preferred embodiment according to the invention relates to a method in which the proteolytic degradation of the Proteins or polypeptides through endosomal and / or lysoso male enzymes.

This embodiment of the method according to the invention is This is particularly advantageous if the antigenic peptides pass through MHC class II molecules are to be presented. How be Already mentioned above, the MHC class II moles not primarily cool antigenic peptides to be loaded by degradation using a proteasome complex, but through proteolytic degradation in endosomal / lysosomal com partitions processed.

In a further preferred embodiment of the invented The method according to the invention derives for the loading antigenic peptide MHC class II molecules to be provided from trans-Golgi vesicles, endosomes or CPLs.

In these vesicles, the MHC class II molecules are so far is known in the art, at least in the case of CPLs least when entering the compartment by the invariante Chain prevented from binding with an appropriate one antigenic peptide. However, it can be assumed hen that in these compartments the proteolytic degradation the invariant chain begins or is completed, so that the MHC class II molecules subsequently with the desired one can associate antigenic peptide.  

In a further preferred embodiment of the invented The method according to the invention is associated with the antigen peptides with MHC class II molecules by common In cubation in CPLs.

With this embodiment of the in vitro The procedure is the in vivo situation of loading the MHC Class II molecules mimicked with antigenic peptides. Therefore should this preferred embodiment of the fiction according to a particular degree lead to the fact that actually interesting antigenic peptides from the MHC Class II molecules recognized and for further investigation can be made available.

In a further preferred embodiment of the invented The method according to the invention is the proteins or polypeptides of natural origin.

For this embodiment it is necessary that a source available on natural protein or polypeptide is sufficient quantities for effective loading charge and presentation by a sufficient number of MHC molecules enables.

In a further preferred embodiment of the invented The method according to the invention is the proteins or polypeptides recombinant or synthetic origin.

This embodiment is particularly preferred since recom binant or synthetic polypeptides with conventional Processes are easier to manufacture than the insulation proteins or polypeptides from natural sources. By that have been developed in the prior art in recent years It is particularly easy to use recombinant proteins in procedures to provide sufficient quantities, which then degraded by the cellular processing machinery and are taken up in large quantities of free MHC molecules can.  

Synthetic polypeptides or proteins are standard process for example using F-moc chemistry a peptide synthesizer from Applied Biosystems Inc. or of a corresponding device can be produced.

In a further preferred embodiment of the invented According to the method according to the number of suitable MHC molecules through in vitro translations and / or transloka ions of MHC molecules in microsomes or trans-Golgi-Ve sicles increased.

In a further preferred embodiment of the invented According to the method according to the invention, the MHC molecules originate from TAP-ne negative cell lines. An example of such a cell line is T2. This cell line and its production is described in Levy et al., op. cit., Salter et al., EMBO J.5 (1986), 943-949, Salter et al., Immunogenetics 21 (1985), 235-246 and De Mars et al., Proc. Natl. Acad. Sci. USA 82 (1985), 8183-8187. According to the technical teaching described there the person skilled in the art will produce further TAP-negative cell lines.

The embodiments described above increase ent neither the relative amount of interest to be mined Protein in the cell or they increase the number of free ones MHC molecules.

So by adding recombinant or synthetic Polypeptides for cellular processing and presentation machinery excess of antigenic peptides associated with endogenous cellular peptides compete, ge create. The amount of free MHC molecules in microsomes can be achieved by in vitro translation and translocation of MHC molecules in microsomes according to known in the art Procedure and using microsome preparations can be increased from TAP-negative cell lines. The latter version Form prevents the MHC molecules from already being present contact with antigens from exogenous sources  Load peptide with endogenous peptides and block them are.

In a further preferred embodiment of the invented The method according to the invention is used to isolate the MHC moles kül-peptide complexes by precipitation with conformation epitope-specific anti-MHC antibodies.

Such antibodies can be polyclonal or monoclonal Be of origin. Procedure for immunoprecipitation with derar antibodies are well known in the art (see e.g. Burgert & Kvist, Cell 41 (1985), 987).

In a further preferred embodiment of the invented The method according to the invention removes the antigens Peptides from the MHC molecules by elution, preferably Acid elution of the peptides from the MHC molecules.

Such processes for elution, preferably for acidification reelution are sufficiently described in the prior art (see Germain and Margulies; loc. cit. and therein zi references).

In a particularly preferred embodiment of the invent The method according to the invention is followed by a peptide series after the elution cleaning step using HPLC or masses spectroscopy.

Such a cleaning step is particularly advantageous if after separation of the antigenic peptides from the MHC molecules found a heterogeneous fraction of peptides becomes. In particular, in the case of processed peptides, the MHC class II molecules bind due to the fact that they are often displaced NH₂ ends a subsequent sequencing of the peptides without prior purification of the relevant antigenic peptides often only hardly possible.

In addition, prior cleaning is also for Peptides separated from the MHC class I molecules that, an advantage.  

Reverse phase HPLC is preferably used as the HPLC method used.

In a further preferred embodiment of the invented The method according to the invention comprises the separation of the antigens Peptides from the MHC molecules identifying Frak ions containing antigenic peptide.

The fractionation of the separated antigenic peptides comes for example, when the separation by Elution takes place.

In a particularly preferred embodiment of the invent The method according to the invention is used to identify the Fractions through a standard lysis test using poly clonal CTL lines that are specific for the protein or polypeptide or that contains the protein or polypeptide pathogens.

Such a standard lysis test is, for example, that in Chromium release processes known in the art; see. e.g. B. Boes et al., Op. Cit.

This embodiment enables the identification of antigenic peptides that are naturally processed and present peptides as far as possible. This is through the use of polyclonal, antigen-specific CTL-Li nien as a detection system for immunogenic and antigenic peptides reached. These CTLs are derived in vitro from peripheral blood Lymphocytes, through contact with autologous, Epstein-Barr Virus transformed B cells (EBV-B cells), which the right Expressing combined antigen in excess.

Another method known in the art, Generating epitope-specific CTLs is stimulation with peptide-pulsed EBV cells.

These CTLs can also be used to search especially subdomi named peptides are used that are just sufficient  be processed accordingly in order to be detected can.

By using preferably recombinant antigen Exogenous sources also become rare on EBV-B cells processed antigenic peptides in combination with MHC molecules len presents that present in vivo in insufficient numbers be activated to activate CTLs.

The activated CTL lines represent the control instance for the authenticity of the identified antigenic peptides with naturally processed and presented antigens.

In a further particularly preferred embodiment of the The inventive method is used to identify the Fractions by one known in the art T cell proliferation test.

In a further preferred embodiment of the invented The method according to the invention is used to determine the amino acid resequence of the antigenic peptides by sequencing under Use of preferably automated Edman chemistry or tandem mass spectrometry.

Both Edman chemistry and tandem mass spectrums trie as electron spray ionization tandem masses spectrometry associated with microcapillary HPLC on the prior art (cf. e.g. Hunt et al., Science 255 (1992), 1261-1264).

In a further preferred embodiment of the invented According to the method according to the invention, the protein or polypeptide originates from a pathogen or corresponds to a protein or poly peptide or part thereof derived from a pathogen is or is a tumor antigen or is part of it. With this embodiment of the method according to the invention can have clinically relevant pathogens or tumor antigens their antigenic peptide components after cellular processing tion to be examined.  

In a further preferred embodiment of the invented According to the method according to the invention, the antigenic peptide is a subdomi named peptide. The advantages of the method according to the invention for the identification of subdominant antigenic peptides already discussed above. This preferred execution Form of the method according to the invention is therefore particularly then usable, and advantageous if such subdomi nante antigenic peptides are to be recognized by the immune system and use its subsequent stimulation to Effectively combat pathogen / tumor antigen.

In a further preferred embodiment of the invented According to the inventive method, the selection of presentable antigenic peptides increased by transfection of a cell line, in which the cellular processing mechanism works and expressing the MHC molecules can encode with TAP molecules the genes, preferably from different species.

The genes encoding TAP molecules are cloned (Levy et al., op. cit., Germain & Margulies, op. cit.) methods known in the art (e.g. hybridization with probes from conserved areas of the gene) other species can be isolated and cloned.

Since it is suspected that peptide binding to TAP is a selective ver process is, by transfection with other TAP-Mo read from different races (being from one certain polymorphism between different human races sen is assumed), from the rat, mouse, hamster, to only to name a few examples, the absolute number of different ones ner peptide binding confirmation increased on TAP molecules. This allows absolutely more different peptides to be used MHC molecules are loaded, which in particular also the Possibilities to identify subdominant antigenic Peptides are increased.

The genes are preferably cloned into expression vectors.

The transfection of TAP-negative is particularly preferred Cell lines with such genes.  

The invention further relates to the use of the antigenic peptides produced according to the method of the invention for the identification of vaccine components against pathogens or tumor cells or from competitive or antagonistic Peptides in autoimmune diseases.

Those identified with the method according to the invention antigenic peptides can, for example, be coupled to a Carrier according to methods known in the art for im immunization against pathogens or for prophylaxis against Tumor growth can be used.

Competitive or antagonistic peptides can be used in the Pro prophylaxis and therapy of autoimmune diseases to stimulate the previously identified autoimmune reactions to displace peptides from binding to MHC molecules. They can also be used for competitive binding to T cell recipes gates can be used without stimulating the T cells.

Claims (24)

1. A method of identifying antigenic peptides comprising the following steps:

• (a) proteolytic degradation by cellular processing mechanisms of exogenously added proteins or polypeptides that contain the antigenic peptides to a peptide size that allows association with MHC molecules;
• (b) association of the antigenic peptides obtained in step (a) with suitable MHC molecules;
• (c) isolation of the MHC molecule-peptide complexes;
• (d) separation of the antigenic peptides from the MHC molecules; and
• (e) Determination of the amino acid sequence of the antigenic peptides obtained in step (d).

2. The method of claim 1, wherein the MHC molecules MHC class I molecules are. 3. The method according to claim 1 or 2, wherein the proteolyti Proteaso degrades the proteins or polypeptides men done. 4. The method according to any one of claims 1 to 3, wherein the MHC class I molecules originate from cellular microsomes. 5. The method according to any one of claims 1 to 4, wherein the Association of antigenic peptides with the MHC class I moles cool by incubating the peptides with microsomes follows. 6. The method of claim 1, wherein the MHC molecules MHC Class II molecules are.   7. The method according to claim 1 or 6, wherein the proteolyti Breakdown of proteins or polypeptides by endoso male and / or lysosomal enzymes. 8. The method according to any one of claims 1, 6 and 7, wherein the MHC class II molecules from trans-Golgi vesicles, Endoso men or CPLs. 9. The method according to any one of claims 1 and 6 to 8, wherein the association of the antigenic peptides with the MHC class II molecules by common incubation in CPLs follows. 10. The method according to any one of claims 1 to 9, wherein the Proteins or polypeptides are of natural origin. 11. The method according to any one of claims 1 to 9, wherein the Proteins or polypeptides recombinant or synthetic are of origin. 12. The method according to any one of claims 1 to 11, wherein the Number of suitable MHC molecules by in vitro trans lation and / or translocation of MHC molecules in Microsomes or trans-Golgi vesicles is increased. 13. The method according to any one of claims 1 to 12, wherein the MHC molecules come from TAP-negative cell lines. 14. The method according to any one of claims 1 to 13, wherein the Isolation of the MHC molecule-peptide complexes by precipitate tation with anti-MHC Antibodies. 15. The method according to any one of claims 1 to 14, wherein the Separation of the antigenic peptides from the MHC molecules by elution, preferably acid elution of the peptides the MHC molecules.   16. The method of claim 14, wherein the elution is a pep tide cleaning step using HPLC or Mas follow spectroscopy. 17. The method according to any one of claims 1 to 16, wherein the Separation of the antigenic peptides from the MHC molecules identifying fractions that include antigenes Contain peptide. 18. The method of claim 17, wherein the identification the fractions using a standard lysis test of polyclonal CTL lines that are specific for the protein or polypeptide or that the protein or pathogen containing polypeptide. 19. The method of claim 17, wherein the identification the fractions by a T cell proliferation test follows. 20. The method according to any one of claims 1 to 19, wherein the Determination of the amino acid sequence of the antigenic peptides by sequencing using preferably automatic Edman chemistry or tandem mass spectrums trie takes place. 21. The method according to any one of claims 1 to 20, wherein the Protein or polypeptide derived from a pathogen, or a protein or polypeptide or a part thereof speaks that comes from a pathogen or a Tu morant or part of it. 22. The method according to any one of claims 1 to 21, wherein the antigenic peptide is a subdominant peptide. 23. The method according to any one of claims 1 to 22, wherein the Selection of presentable antigenic peptides increased  is by transfection of a cell line in which the zel lular processing mechanism expires and the MHC-Mo can express with Ge encoding TAP molecules NEN, preferably from different species. 24. Use of one according to one of claims 1 to 23 ago provided antigenic peptide for the identification of Vaccine building blocks against pathogens or tumor cells or of competitive or antagonistic peptides Autoimmune diseases.